A wide variety of devices are used to study the property of materials. For example, confocal microscopes are often used to study the spectral characteristics of materials responsive to being excited by electromagnetic radiation, such as a laser pulse. In a conventional confocal microscope, light from an excitation laser is directed through an objective lens of the microscope, which focuses the light on a sample. Fluorescence from the sample is collected and imaged through a pinhole to eliminate light that does not originate from the focus of the laser in the sample. This improves the spatial resolution of the microscope. The fluorescence light that passes through the pinhole is detected and the signal recorded. To form an image of the sample, either the sample is scanned through the laser or the laser is scanned across the sample while the resulting fluorescence is detected and the signal recorded. In conventional confocal microscopes, structures in the (usually biological) sample are stained with fluorescent dyes. Dyes with different fluorescence characteristics have been developed along with methods of specifically attaching these dyes to particular structures. Identifying the dye, usually through the wavelength range of its fluorescence, thus identifies structures in the sample. Characteristics of the fluorescence such as the lifetime and spectrum contain additional information about the sample and distinguishing them can provide additional contrast in the image. An important challenge in applying confocal microscopy is the need to obtain as much information from the fluorescent dyes as possible before the dyes bleach from exposure to the exciting laser light. Conventional photon detectors are often incapable of providing sufficient information before excessive bleaching of the dyes has occurred.
A sample exposed to a moderate level of excitation, such as a laser pulse, may not emit a photon responsive to each laser pulse. Instead, the sample may emit a photon infrequently, such as one every 50 laser pulses. However, there is a finite probability that a photon will be omitted responsive to each laser pulse. The probability of emitting a pulse as a function of emission delay time is known as the “fluorescence lifetime.” A typical fluorescence lifetime graph is shown in FIG. 1 in which the probability “P” of emitting a photon is plotted on the Y-axis and the time delay “τ” between the excitation and the emission of a photon is plotted on the X-axis. As shown in FIG. 1, the probability of emitting a photon decreases as a function of time after the excitation pulse. FIG. 1 shows that, if the excitation pulse results in a photon being emitted, the photon is most likely to be emitted soon after the excitation pulse. Conversely, if a substantial time has lapsed since the excitation pulse without a photon being emitted, there is relatively little probability that a photon will be emitted from the sample at all.
In the past, it has been fairly difficult and time consuming to obtain sufficient data about a sample to determine its fluorescence lifetime and the wavelengths of its emitted photons. This difficulty is primarily due to the very small delay time between the excitation pulse and the emission of a photon. Typical delay times are on the order of 2-3 ns (10−9 sec.), and delay time measurements should be made with resolutions on the order of 10-20 ps (10−12 sec.). It can be very difficult to measure time periods of such small durations. The difficulty in obtaining sufficient data also results from the relatively few number of excitation pulses that result in a photon being emitted, coupled with the need to obtain data about a large number of emitted photons. Data for a large number of photons must be collected because the probability of emitting a photon at each delay time is determined by counting the number of photons emitted at each delay time. A larger sample provides more accurate results. As a result, the sample must be exposed to a very large number of excitation pulses to emit enough photons to make an accurate determination of fluorescence lifetime.
There are situations where the fluorescence lifetime depends on the wavelength. This relation between lifetime and wavelength will only be observed if the emission time and wavelength of each photon are simultaneously measured. The correlation between lifetime and wavelength of the fluorescence can supply additional information about the sample being observed. Various approaches have been used to determine the wavelengths and fluorescence lifetimes of photons emitted from samples. For example, photons emitted responsive to laser pulses have been coupled to photodetectors through bandpass filters that allow photons to pass only if they are within one or more narrow bands of wavelengths. However, this approach can provide data only if the wavelength(s) of the emitted photons is known. It may not provide accurate results if the wavelengths are not known, nor can it easily determine the wavelength of emitted photons is the photons are emitted at a large number of different wavelengths, such as an entire spectrum of wavelengths.
To detect changes in a sample, such as those caused by diffusion, it is important to obtain the fluorescence spectral and lifetime information rapidly compared to these changes. In practical situations this often requires the detection system to be capable of recording photons at rates of 1×106 photons/second or faster.
There is therefore a need for a device and method that is capable of simultaneously measuring the wavelengths and delay times of emitted photons, and doing so in a quick and easily manner, and in a manner that provides sufficient information about the sample before fluorescent dyes used in the sample have been excessively bleached.